In addition to their essential role in the elicitation of immediate hypersensitivity reactions, mast cells have been implicated in the innate immune response to infectious bacteria. We found that human mast cells bind and internalize E. coli as assessed by both flow cytometric analysis and confocal microscopy. This process results in up-regulated activation-dependent gene expression by human mast cells as analyzed by DNA microarray assays. Transcripts up-regulated include those coding for enzymes, surface molecules, components of the cytoskeleton and molecules associated with signaling, the cell cycle, adhesion, and protein transport. More specifically, and relevant to innate immunity, genes encoding for TNF-alpha and three chemokines of the CC family e.g., I-309, MIP-3, and MIP-4 were found to be over-expressed following exposure of human mast cells to bacteria. We have analyzed Fyn-binding proteins in MC/9 mast cells to explore the Fyn-mediated signaling pathways, and identified 6 proteins that bind to Fyn including vimentin, pyruvate kinase, p62 ras-GAP associated phosphoprotein, SLP-76, HS-1, and FYB. After IgE-receptor mediated stimulation, binding of vimentin to Fyn was increased. Mast cells from vimentin-deficient mice showed enhanced mediator release and tyrosine-phosphorylation of intracellular proteins including NTAL and LAT.